Screening the nematicidal potential of indigenous medicinal plant extracts against Meloidogyne incognita under lab. and greenhouse conditions

The root-knot nematode, Meloidogyne incognita, causes a high damage and yield decrease for many economic plants. The need for non-systemic effective new approaches and environmentally friendly methods for controlling the nematodes has directed research to some new and safe agrochemicals found in medicinal plants as new viable management options. In laboratory experiments, solidago and periwinkle aqueous and ethanolic extracts achieved high J2 mortality (%) concerning different dilutions; however, aqueous extracts were more effective for mortality than ethanolic extracts. Also, there was a direct relationship between the nematicidal activity of these extracts with both concentration and time of application. Inhibition of egg hatching by Periwinkle extracts was higher than that of solidago. Moreover, the nematicidal activity of tested extracts against J2 decreased significantly with prolonged storage time at + 5 °C, while did not with stored frozen at – 5 °C for 12 months. Periwinkle and solidago extracts killed the non-target organisms, i.e., rotifers and free-living nematodes. Seventy-five and 90% of total phytochemicals recovered from periwinkle and solidago, respectively were nematostatic or nematicidal to nematode viability, egg hatch in vitro, and development and reproduction in vivo despite the method of application (foliar and soil drench). The antagonistic effects of solidago were more pronounced in soil drench than periwinkle concerning their concentrations and methods of application. Solidago and periwinkle plant extracts showed important sources of effective control phytochemicals against M. incognita.


Background
Botanicals can be used in in vitro and in vivo by different ways as one of the nonchemical approach strategies to manage and reduce plant-parasitic nematodes, especially in sustainable agriculture (Bridge 1996) by using their parts directly, their extracts, and compounds that possessing nematicidal activities, oilseed cakes, mature crop residues as organic amendments (Manju and Meena 2015). Some of the botanicals are already being exploited commercially in pest management and a rising trend towards organic farming (Zaidat et al. 2020).
In vitro, a lot of plant extracts showed high ovicidal and nematicidal effects on egg hatching and J2 survival of the root-knot nematode (RKN), M. incognita. Extracts from Nicotiana tabacum, Syzygium aromaticum, Piper betle, and Acorus calamus were found more effective in killing M. incognita, with an EC 50 which was 5-10 times lower than the EC 50 of the synthetic pesticides, chlorpyrifos, carbosulfan, and deltamethrin (Taniwiryono et al. 2009). The nematicidal effect of plant extracts could be higher than synthetic nematicides.
In vivo, (under greenhouse or field conditions) application of plant extracts reduced infection of RKN nematode and caused crop yield increase. These extracts were more effective than the nematicides used or in the same order or slightly less.
Dozens of phytochemical compounds that may be more active and eco-friendly, especially those came from medicinal and aromatic plants, e.g., serpentine, saponins, phenols, alkaloids, tannins, flavonoids, steroids, and cysteine proteinases have been reported for their antihelmintic effect against human, animal, and plant parasites (Rocha et al. 2017). The nematicidal effects of dried parts and boiled extract of Bidenspilosa were bioactive when re-evaluated on phytoparasitic nematodes after storing up to 12-18 months (Taba et al. 2012).
This study focused on the nematicidal potential of some medicinal plant extracts in the management of root-knot nematode in vitro and in vivo and their preservation on nematicidal activity, non-target organisms, and chemical composition.

Nematode culture
Pure stock culture of the RKN, M. incognita originally obtained from galled eggplant roots was established. Single egg-mass from previously identified females (Taylor et al. 1955) was used to inoculate healthy eggplants grown in (20 cm diameter) earthen pots filled with loamy sand soil. Three months after inoculation, plants were examined for nematode infection and reproduction. The culture was propagated and maintained on eggplant.

Plant extracts preparation
The effect of aqueous and ethanolic extracts of 13 medicinal plants was evaluated directly or after storage periods for lethal concentrations and toxicity index on survival and hatchability of M. incognita and non-target organisms' management under laboratory conditions (in vitro). Therefore, foliar spray and soil drench applications of solidago and periwinkle, which achieved the highest mortality percentages than the other tested plant species were carried out in the greenhouse (in vivo) on infected sunflower plants.

Aqueous extracts
Twenty-five grams of air-dried leaves of 13 medicinal plants listed in Table 1 were homogenized by grinding for 1 min using a blender to coarse particles (formation of extremely soft particles like a powder that may hamper better extraction was avoided by the solvent as described by Pandey and Tripathi 2014). Then, 1 L of tap water was added. The mix was transferred to a 2-L beaker and was shaken vigorously. Decoction process was done to extract watersoluble and heat-stable constituents, in which the mix was boiled for 10 min, cooled, and filtered using filter paper. This mix was kept frozen as a stock solution (1×) until use. The stock solution was diluted by adding tap water to prepare the diluted extracts. Seven extracts dilutions (1:2×, 1:4×, 1:8×, 1:16×, 1:30×) were prepared by adding sufficient tap water to the stock solution till reaching the required concentrations. The 5 dilutions were equivalent, respectively, to the 5 concentrations (12500, 6250, 3120, 1560, 780 mg dry weight/liter (mg D.Wt./L)).

Ethanolic extracts
Five grams of the previously mentioned medicinal plants' air-dried leaves were homogenized to coarse particles using a blender, then added to 200 ml of ethyl alcohol 96% in a 1-L beaker, shaken for 24 h using a shaker at room temperature, and filtered using a filter paper. A rotary evaporator was used to evaporate the solvent (ethanol) under vacuum to prepare the crude extracts, which then were dissolved in 5 ml ethanol and added to 200 ml tap water + 1 ml tween 80 as a surfactant. The resulting solution was shaken and kept frozen as a stock solution (1×). This stock solution is equivalent to a concentration of 25 mg D.Wt./L. Only 4 dilutions (1/2×, 1/4×, 1/8×, and 1/16×) were prepared by adding tap water to the stock solution till reaching the required dilutions, equivalent to 12500, 6250, 3120, 1560 mg D.Wt./L concentrations, respectively. Calculating lethal concentrations and toxicity index for M. incognita J2 Data of mortality percentages (%) in vitro were input to LDP line software to calculate probit analyses according to Finney (1971), which was used to illustrate the relation between stimulus and response in toxicological studies. The toxicity index of each plant extract was determined according to Sun (1950) using the following formula: Effects on non-target organisms Concentrations of 1000 and 2000 mg D.Wt./L of the periwinkle and solidago extracts were tested against 2 kinds of metazoans; Rotifers and free-living nematodes in vitro. Four replicates for each treatment were set and mortality percentages were calculated after 48 h. Each replicate contained a mix of approximately 80 free-living nematodes and 100 rotifer individuals. The population of free-living nematodes was obtained from a soil sample rich in organic matter. However, rotifers were obtained from a soil sample kept at room temperature for a month to increase rotifers counts. The mixed population in water was kept as a check.

Storage periods of aqueous extracts
Samples of freshly prepared stock of each of solidago and periwinkle leave extracts were stored either under freezing at -5°C for 1 year, cooled for 2 weeks or 2 months at + 5°C, then they were re-evaluated for their nematicidal effect changes. The experiment procedures and mortality percentages were done as previously mentioned on M. incognita J2 in vitro.

Greenhouse experiments
Based on data obtained from the mortality in vitro tests on M. incognita J2s, solidago and periwinkle aqueous extracts were tested under greenhouse conditions for M. incognita control. Seeds of sunflower (Giza-102) plants were sown in 220 pots, each filled with 2 kg sandy clay soil (1:1, v:v). Two weeks later, pots were divided into two main groups which were treated as follows:

Foliar spray application
The aqueous extracts were applied as a foliar spray once. Foliar spray drift to the soil was avoided by covering it, using a tissue paper. The tested plant aqueous extracts concentrations were 500, 1000, and 2000 mg dry leaves/L (mg D.Wt./L). Each treatment contained 5 replicates. After 2 weeks of germination, plants were inoculated with 2000 M. incognita J2s/ plant. The whole experiment was set and horticultural-maintained for 45 days after nematode inoculation. Only one factor was different for each of the 3 sub-groups, which was the time application, as follows: the first subgroup; extracts were foliar sprayed 1 week before nematode inoculation, the second sub-group; extracts were foliar sprayed simultaneously with nematode inoculation, and the third subgroup; extracts were foliar sprayed 1 week after nematode inoculation. Five replicates in each subgroup received the same treatments, except that instead of applying plant extracts, the synthetic pesticide formulation, Vydate® 24% SL (oxamyl) was foliar sprayed as a standard chemical nematicide (3 ml/L). Another 5 replicates were inoculated with nematode only and kept as a check.

Soil drench application
The aqueous extracts were applied as a soil drench once (100 ml/plant). The tested plant aqueous extracts (solidago and periwinkle dry leaves extracts) concentrations were 500, 1000, and 2000 mg dry leaves/L (mg D.Wt./L). This group contained 110 pots, each treatment contained 5 replicates. Inoculum level was 2000 J2 of M. incognita after 2 weeks of germination. One factor was different for each of the 3 sub-groups, which was the time of extracts application as follows: the first subgroup; 100 ml of extracts solution were applied as a soil drench for each pot 1 week before nematode inoculation, the second sub-group; 100 ml of extracts were applied simultaneously with nematode inoculation, and the third sub-group: 100 ml of extracts were applied 1 week after nematode inoculation. Five replicates in each subgroup were received the same treatments except that, instead of applying plant extracts, the synthetic pesticide formulation, Vydate® 24 % SL, was soil drenched as a standard chemical nematicide (0.1 ml/L). Another 5 replicates were inoculated by nematode only and kept as a check.

Nematode assay
Upon harvest, each pot was soaked in a plastic bucket filled with water until the root system could be easily separated. Each root system was weighed and stored in 5% formaldehyde in plastic jars. The soil suspension was quite stirred and then poured through a series of 60 and 325 mesh screens (Hooper et al. 2005). The bottom sieve was then poured onto a modified Baermann set and collected after 48 h. Hawksley counting slide was used to calculate the number of J2s in 1 ml of the suspension and then referred to the whole volume. The numbers of galls and egg-masses were counted directly on the root system of each replicate and the mean of each treatment was calculated and later the eggs were extracted (Boneti and Ferraz 1981). For calculating eggs per egg mass, 10 full egg masses from each replicate were chosen, gelatin matrix was dissolved using sodium hypochlorite (NaOCl) according to (Hussey and Barker 1973), and eggs were counted in 1 ml volume under the microscope. The final population (eggs + soil population) plotted in the formula RF = Pf/Pi, where RF is the reproduction factor, Pf the final population, and Pi the initial population (Oostenbrink 1966).

GC/MS/MS analysis Sample preparation
Five grams of grinded dried leaves of each plant (periwinkle and solidago) were added to 100 ml deionized water in a 250-ml flask. The decoction process was done as previously mentioned. The extract was filtered, using filter paper, centrifuged at 9000 rpm for 5 min to exclude any impurities, then it was lyophilized (freezedried).

Chromatographic analysis
Produced powder from the last step was analyzed using gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry (GC/MS/MS).
The analysis was carried out using a GC (Agilent Technologies 7890A) interfaced with a mass selective detector (MSD, Agilent 7000) equipped with a polar Agilent HP-5ms (5%-phenyl methyl polysiloxane) capillary column (30 m × 0.25 mm i. d. and 0.25 μm film thickness). The carrier gas was helium with a linear velocity of 1 ml/min. The injector and detector temperatures were 200 and 250°C, respectively. The volume injected 1 μl of the sample. The MS operating parameters were as follows: ionization potential 70 eV, interface temperature 250°C, and acquisition mass range 50-800. The identification of components was based on a comparison of their mass spectra and retention time with those of the authentic compounds and by computer matching with NIST and WILEY library as well as by comparison of the fragmentation pattern of the mass spectral data with those reported in the literature (Dong et al. 2014).

Statistical analysis
Data were statistically analyzed one-way ANOVA according to the SPSS software package version 23. The differences between means were tested using Duncan's multiple tests at the 5% significance level.

Effect of aqueous extracts on J2 mortality
Dry leaf powder extracts of 13 medicinal plant species were tested in a laboratory assay on M. incognita J2s' mortality with 5 dilutions of each. In general, after 48 h of exposure, all extracts used had nematicidal action against M. incognita J2 depending on the plant species and rate of the aqueous extract dilution ( Table 2). The percent of mortality decreased by increasing extracts dilutions, except that of solidago and periwinkle dry leaf extracts. However, the first 2 dilutions 1:2× and 1:4× were highly toxic in all extracts achieving 100% mortality. Leaf extracts of solidago and periwinkle displayed the highest toxicity in (1:16× and 1:30× dilutions) than the rest of medicinal extracts, although, the percentage of mortality decreased down to almost 70% at (1:30× dilution). A considerable decrease in J2 mortality appeared by (1:16× dilution), less than 50% of J2s were killed by Common mint, horsemint, geranium, and dill extracts. The least percentages of mortality at (1:30× dilution) were recorded by extracts of marjoram, chamomile, and geranium, which were almost similar to the acceptable natural death percentage in the check (without treatment).
The relative toxicity index was measured by calculating and comparing each J2's mortality relative to the maximum percentage of mortality. The accumulative percentages of mortality were calculated as well and were the highest in periwinkle followed by solidago and the least was found in Fros dill. The toxicity index, as well as the accumulative percentage of mortality, followed the same trend where the highest toxicity index was gained by solidago extract, followed by periwinkle and marjoram, respectively. While dill recorded the lowest relative toxicity value.
Concerning the lethal concentration needed to achieve LC 50 , it was found that solidago recorded the lowest lethal concentration that achieved such criteria and it was almost 1/2:1/3 of most extracts concentrations, followed by periwinkle. Geranium and dill needed four times concentrations as much as solidago to attain LC 50 . LC 90 was found to be achieved by a concentration of almost 1 g D.Wt./L in solidago extract. The lowest lethal concentration for LC 90 was raised to 5 times in horsemint, 10 and 11 times in rosemary and thyme extracts, respectively. Solidago and periwinkle extracts recorded almost the same lethal concentration required to achieve LC 90 .

Effect of ethanolic extracts on J2 mortality
The ethanolic extracts of the previously mentioned medicinal plant species were tested on M. incognita J2s mortality at 3 dilutions. After 48 h exposure, the ethanolic dry leaf extracts of all medicinal species were found lethal to M. incognita J2s, regardless of the rate of dilution, although the mortality percentages differed according to plant species extract and dilution (Table 3). There was direct proportional relation to a great extent between mortality and dilution. Considerable high mortality rates were noticed at 1:2× dilution in most cases than 1:4× or 1:8× dilutions. Coriander, geranium, solidago, marjoram, basil, and periwinkle extracts achieved higher mortality in 1:4× dilution than that of 1:2× or 1:8× dilutions. The general decrease in J2 mortality was recorded in 1:8× dilution, when compared with the others. The accumulative mortality percentages of geranium, marjoram, and solidago were the highest meanwhile; horsemint, chamomile, and dill were the lowest. However, the toxicity index of solidago extract was in the lead of all extracts.
From the results, it seems that the aqueous extracts of dry leaves of medicinal species were much more efficient in being nematoxic or nematicidal to M. incognita J2s than ethanolic extracts in all dilutions and that was reflected positively on the least lethal concentrations required for LC 50 and LC 90 .

Effect of storage aqueous extracts against J2
Results of the previous in vitro experiment verified that extracts of solidago and periwinkle recorded the ultimate percentages of J2 mortality amongst all the tested aqueous extracts. So, the effect of storage conditions and temperature on extracts stability and activity and in turn, their immobilization activity was studied (Table 4). The storage time affected the extract's capability on mortality. The nematoxic effects of extracts were match able to those of expected fresh after 14 days storage in the fridge at + 5°C, but when the storage period was prolonged up to 60 days, the stability of the phytochemicals was drastically affected, which in turn minimized mortality in solidago aqueous extract treatments. That minimization was more pronounced at 1000 mg D.Wt./ L concentration. The activity of periwinkle extract lower concentration was less effective than the higher one. The activity of extracts differed after 12 months of freezing conditions at -5°C. Periwinkle activity was stable achieving 92% mortality at 1000 mg D.Wt./L and heightened up to 100% at the higher concentration. The opposite was found to be the case in solidago extract lower concentration (78% mortality). The higher concentration was highly effective as much as expected fresh or 14 days cooled storage. It seems that J2s mortality retrogrades proportionally with the time of extract storage.  The efficacy of treatment was improved by doubling the concentration but not with the time of storage.

Effect of aqueous extracts on egg hatching
Solidago and periwinkle aqueous extracts which were found to be the most efficient on M. incognita J2s mortality were selected to study their effect on hatching of egg masses at 3 concentrations after 7 days of exposure. Significant inhibitory effects on the numbers of hatched eggs when compared with the check were observed (Fig. 1). Also, significant differences were found between and within treatments. Periwinkle extract was significantly efficient in reducing the number of hatched eggs at all concentrations. Non-significant differences were found between the lowest and middle concentrations of periwinkle. There was a significant positive correlation between the concentration of each aqueous extract and the rate of inhibition. The highest rate of inhibition was recorded at periwinkle (98.9%), followed by solidago (87.5%) with the highest concentration (1000 mg D.wt./ L). The lowest inhibitory effects were found in the case of the lowest concentration of solidago extract (23.4%).

Effect of periwinkle and solidago aqueous extracts on non-target organisms
A further in vitro experiment was conducted to study the effect of solidago and periwinkle dry leaf aqueous extracts at 2 concentrations (1000, 2000 mg D.Wt./L) on the free-living nematode genera and rotifers, which were found co-inhabiting the soil with the root-knot nematodes. The recessive effects on both microorganisms were observed regardless of the type of plant extract as well as concentration; however, solidago extract was more efficient than periwinkle were obvious in Fig. 2. Effects on rotifers were more retrograded than free-living nematodes. Mortality of rotifers after 48 h treatment was consistent in periwinkle leaf extract at the two used concentrations, which achieved more than 50% mortality. It is interesting to notice that percentage of mortality in solidago treatments was directly correlated with concentration, 66.5 at the lower and 84.7 at the higher. On the other hand, solidago aqueous extract interpreted almost similar mortality percentages at the lower and higher concentrations on free-living nematodes. Periwinkle extract at 1000 mg D.wt./L recorded mortality values on free-living nematodes 10% less than its higher concentration which was close to solidago extract concentrations.

Chemical content of solidago and periwinkle dry leaves
Quantitative and qualitative analysis of phytochemicals in dry leaves of periwinkle and solidago was carried out by GC-MS-MS. The leaf extract manifested four main phytochemical classes. Flavonoids, terpenoids, phenolics, and coumarins were highly present with different concentrations. Flavonoids were the most dominant in all the extracts, followed by the terpenoids, phenolics, and coumarins in the solidago leaf extract. Phenolic compounds were the maximum class found in periwinkle leaves (59.2%), followed by flavonoids (19.7%) and coumarins (5.2%). The minimum was piperazines (3.9%), glycosides (2.6%), and phytosterols (2.4%) in solidago leaves, respectively. Meanwhile, the lowest ratios were organic alcohol (4%), fatty acids (4.1%), alkaloids (3%), and sesquiterpenoids (3%) in periwinkle leaves. There were correspondingly prohibiting effects of the main chemical classes and the ratios on nematode activity, egg inhibition and development, and reproductively.

Effect of periwinkle and solidago aqueous extracts on RKN infecting sunflower
Potentials of periwinkle, solidago dry leaves aqueous extracts under 3 intervals (pre, with, and post-inoculation), 2 methods of application (foliar spray, soil drench) with 100 ml of each of the concentrations (500, 1000, 2000 mg D.Wt./L), and the comparable nematicide Vydate® 24% SL on M. incognita in greenhouse experiment was detected and illustrated in Tables 5 and 6.

Foliar spray application
Apropos of pre-inoculation, periwinkle, and solidago extracts significantly suppressed the numbers of formed galls, egg masses, and eggs/egg mass. Except for periwinkle extracts, the numbers of eggs exceeded the nematode check and consequently reflected on nematode reproduction, which was more or less the nematode check. Solidago treatment concentrations performed significant impressive reductions in all nematode criteria and exhibited smashup in nematode final population and buildup. Obvious direct proportional effects were noticed among solidago concentration. Very few eggs were laid in the highest sprayed concentration (2000 mg D.Wt./L) achieving the lowest buildup (Table 5).
As for inoculation abreast with foliage spray, all treatments concentration imposed significant smashing reductions on all nematode criteria either intra-or interspecific treatments except the foliar sprays of solidago at 500, 1000 mg D.Wt./L at the number of deposited eggs and buildup, where the nematode was able to fold only once with non-significant differences with the check. Periwinkle concentrations were the most invincible where the least buildup rates were performed.
The post-inoculation treatment followed the same trend with the abreast one, all treatments were significantly effective in reducing numbers of galls, egg masses, final population, and the subsequent buildup and egg production when compared with the check. Solidago and periwinkle at 1000, 2000 mg D.Wt./L outmatched Vydate® 24% SL.
Concerning the foliar spray treatment, Vydate® at all intervals, the nematode galls, egg masses, and fecundity were sharply declined. Values of buildup were eliminated with significant differences where the nematode was not able to fold even once in pre-and post-inoculations.

Soil drench application
Efficacies of the aqueous extracts of periwinkle and solidago on M. incognita at different intervals and same concentrations as previously mentioned and treated as a soil drench to sunflower plants are given in Table 6. It contended in general that soil drench applications were more effective in reducing nematode criteria than foliar spray as measured by gall formation, egg masses fecundity, and buildup. Referring to the used concentrations, most if not all extracts concentrations were highly suppressive for nematode parameters. Solidago extracts surpassed to great extent periwinkle extracts at all intervals and concentrations. Controvert effects were noticeable in periwinkle extracts at 500, 2000 mg D.Wt./L at preand post-inoculation intervals, where the nematode was able to fold more than once as compared with the untreated inoculated check. The detersive effects of Vydate® were more malignant when treated as soil drench than foliar spray and in particular when treated, with inoculation, no J2s were able to invade the roots.

Discussion
GC/MS/MS analysis indicated the presence of phytochemicals belonging to the following classes: phenols, flavonoids, triterpenoids, coumarins, alkaloids, and glycosides. groups with different concentrations. Solidago and periwinkle extracts were in the lead of all the 13 tested extracts in achieving the highest percentages of mortality. Ethanol extracts were effective in increasing mortality to M. incognita J2s but not as much as aqueous extracts. This may be due to the polarity of the solvent; the polarity of water is higher than that of ethanol. Consequently, higher antioxidants and phenols were extracted using water. Ng et al. (2020) reported that polar solvents could extract higher amounts of antioxidants and phenolic compounds, which increase the radical scavenging activity of the extracts. Neeraj et al. (2017) reported that alcoholic extracts showed a high activity in immobilization of M. incognita J2s and egg hatching inhibition in vitro. That may be because they used high concentrations. The nature of medicinal plant structure and their derivatives have been discussed extensively but the mode of action of most nematicidal phytochemicals is still ambiguous. Periwinkle achieved more inhibitory effects on egg hatchability than solidago extract. That inhibition was proportionally correlated with concentration increase. It is well known that periwinkle extracts are rich in alkaloids. Extracts that contain alkaloids were found to have ovicidal property against Meloidogyne eggs (Adegbite 2003). Also, Alkaloids may act on the central nervous system and cause paralysis (Roy et al. 2010), which gave nematicidal effects. Important notice should be considered when comparing the efficacy of ethanolic and water extracts; solidago extracts with both solvents were the most toxic on J2. However, Periwinkle extract nematicidal effects were pronounced only in the aqueous extract and reduced greatly in the ethanolic one. Herein, the nematicidal and ovicidal effects of periwinkle extract are basically due to the water-soluble fractions.
Solidago aqueous extract had a higher percentage of compounds known with their nematicidal activity than periwinkle extract; such as terpenoids (Ohri and Pannu 2009), glycosides (Pronar 1983), and piperazines. Actually, for a long time, piperazine derivatives are used as antihelmintic drugs for humans (Shafei et al. 1955).
There is a real need for fractionation of the constituents of the extract to test each compound individually. However, one can generalize that the bioactivity against nematodes of each extract follows a multi-site mode of action. This is simply because that there is a large number of compounds in each extract, and these compounds have different functional groups with different modes of action. Flavonoids, low molecular weight secondary metabolites have diverse functions including defense and auxin transport inhibition, and are implicated in resistance to both sedentary and migratory nematodes (Baldridge et al. 1998). Also, hypersensitive response and accumulation of the phytoalexin glyceollin, a product of isoflavonoids inhibit oxidation, respiration of M. incognita in vitro and accumulate adjacent to the head region of soybean cyst nematode in resistant root tissues (Kaplan and Keen 1980). Phenolic compounds interfere with the energy generation mechanism by uncoupling the oxidative phosphorylation and interfere with glycoprotein of the cell surface of the parasite and cause death (John et al. 2009). Glycosides nematicidal effects are due to their function as a cholinesterase inhibitor that prohibits the normal buildup of nematode (Pronar 1983).
It has been found that prolonging the time of storage of extracts affects their stability and minimizes activity on nematode mortality, which was also influenced by the extract type. Aqueous extracts of periwinkle and solidago could be stored for 12 months without significant loss of nematicidal activity against M. incognita. The boiled extracts of Bidenspilosa could be stored for 12-18 months without loss of nematoxic activity (Taba et al. 2012).
Periwinkle and solidago extracts showed toxic effects on the free-living nematode and rotifers in vitro. Logically it may be lower than that of the synthetic nematicides but plant extracts are still considered xenobiotics and could alter the normal activity of the microfauna in the soil. Storage experiment revealed that bioactive compounds in these extracts were stable only at freezing temperatures, so biodegradation of these extracts in the soil is expected in a short period.
The greenhouse results emphasized the toxicity action of periwinkle and solidago extracts either foliar spray or soil drench treatment under different intervals on M. incognita parameters. The negative effects of solidago were more pronounced than periwinkle in most concentrations, method as well as the time of application. The nematicidal phytochemicals represented 90% of the total phytochemicals recovered from solidago meanwhile, that percentage was 75% for periwinkle. The nematode reduction outcomes may be due to the toxic contact action to the nematode juveniles and to the small molecular weight phytochemicals, which absorbed by roots easily that raise the plant resistance against nematodes or may have systemic nematicidal action as proven by pre-, with, and post-inoculation applications. That systemic nematicidal action may be attributable to the high ratios of phytochemical components (Archana and Parasad 2014) on other plants with different concentrations. Flavonoids could help in plant resistance against nematodes by affecting chemotaxis towards roots and interfere with functions in nematode reproduction (Chin et al. 2018). They added that the mechanism of these effects is still unknown. This can interpret the decline in reproduction factor and number of galls due to the application of both extracts. The medicinal plant extractions treated as soil drench showed significant reducing effects and nematicidal activity to M. incognita criteria (El-Nagdi and Youssef 2013). The described effects on M. incognita were achieved by using plant extracts with low concentrations under lab and greenhouse conditions which started with 0.5 g/l and did not exceed 2 g/l of the dry leaves. Comparing the used concentrations with the chemical nematicides may minimize the coast effectiveness. Solidago and periwinkle might be promising candidates of nematode management tactics.

Conclusion
Extracts of solidago and periwinkle leaves might be promising sources of phytochemicals that have nematicidal activity.